3 replies to this topic

[kertenkele]

Hi everyone,

I have a problem concerning my research design: I want to analyze the effect of exon methylation of a gene in THP-1 cells. The problem is that I have to transfect the THP-1 cells with plasmid containing the methylated gene, however, as you know, I cannot amplify the gene with its methylated state in order to insert it in the plasmid.

Do you have any suggestions? How can I conserve the methylation state of my gene?

Thank you very much in advance.

[Michaelro]

kertenkele, on Feb 15 2010, 12:33 AM, said:

Hi everyone,

I have a problem concerning my research design: I want to analyze the effect of exon methylation of a gene in THP-1 cells. The problem is that I have to transfect the THP-1 cells with plasmid containing the methylated gene, however, as you know, I cannot amplify the gene with its methylated state in order to insert it in the plasmid.

Do you have any suggestions? How can I conserve the methylation state of my gene?

Thank you very much in advance.

Hi
Maybe You should clone your exon into the plasmid vector, prepare the sufficient amount of the resulting plasmid and then methylate it in vitro with M.SscI.
Afterwards, You transfect this methylated plasmid into the cells.
As control, you may use methylated plasmid vector without insert.
In any case, I'm sure that You may find in literature the exact method for transfecting methylated plasmids.
Best
Michael

[kertenkele]

Michaelro, on Feb 15 2010, 10:15 AM, said:

kertenkele, on Feb 15 2010, 12:33 AM, said:

Hi everyone,

I have a problem concerning my research design: I want to analyze the effect of exon methylation of a gene in THP-1 cells. The problem is that I have to transfect the THP-1 cells with plasmid containing the methylated gene, however, as you know, I cannot amplify the gene with its methylated state in order to insert it in the plasmid.

Do you have any suggestions? How can I conserve the methylation state of my gene?

Thank you very much in advance.

Hi
Maybe You should clone your exon into the plasmid vector, prepare the sufficient amount of the resulting plasmid and then methylate it in vitro with M.SscI.
Afterwards, You transfect this methylated plasmid into the cells.
As control, you may use methylated plasmid vector without insert.
In any case, I'm sure that You may find in literature the exact method for transfecting methylated plasmids.
Best
Michael

Thank you Michael, but my problem is more complex I have to clone my entire gene (with its introns) to study other factors like alternative splicing, histone binding sites, etc. and I don't want to methylate the promoter (maybe I can use another promoter without CpG islands)

[tobikenobi]

....sorry to go a little off-topic, but its a just a short one:

Does freezing the cells at -80 before DNA prep and further processing for methylation analysis affect the outcome of the analysis?

Thanks a lot!!!
Tobi